Post-translational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CaaX sequence motifs. Isoprenylation is generally accompanied by two subsequent processing steps, proteolytic cleavage of the aaX residues and carboxymethylation of the newly exposed carbonyl group of the modified cysteine residue. Some isoprenylated proteins also undergo additional proteolytic processing, including an additional cleavage by the same protease that initially removes the aaX residues. As substrates for CaaX processing include ras and rho, small GTPases frequently mutated in cancers, the enzymes of the CaaX pathway are validated targets for cancer drug discovery. Due to its role in maturation of the a-factor mating pheromone, the first-identified and best-characterized CaaX protease is the yeast zinc metalloprotease Ste24p. A human ortholog of Ste24p, ZMPSTE24, can complement the full function of yeast Ste24p. The only known substrate for the human protein is prelamin A, the precursor to the nuclear intermediate filament protein lamin A. Mutations in either ZMPSTE24 or the processing site of prelamin A are associated with a spectrum of premature-aging diseases referred to as progeria. Also, ZMPSTE24 is inhibited by antiviral drugs designed to target the HIV aspartyl protease, and this off-target effect may give rise to some of the severe side-effects of these drugs. We determined the crystal structure of yeast Ste24p. Its core structure is a ring of seven transmembrane helices enclosing a large (14,000 3) interior volume (CAVITY) that contains the active-site and substrate binding region (GROOVE). The cavity is accessible to the external milieu via gaps (PORTALS), partially occluded by non-transmembrane domains, that are between pairs of splayed transmembrane helices. Human ZMPSTE24, solved contemporaneously by another group, possesses the same architecture. The structures of yeast Ste24p and human ZMPSTE24 are quite similar, with a pairwise RMSD of ~1.2 . We propose that cleavage proceeds via a processive processing mechanism (PPM) of substrate insertion, translocation and ejection. The role of the large cavity, the mechanism of specific recognition of cleavage sites with divergent sequences, the unusual dual cleavage process, and the role of the farnesyl group in recognition are some of the questions we seek to address with this research proposal that features structure-function studies of the yeast and human enzymes.